The Allosteric Regulation of Pyruvate Kinase

Abstract

form an intersubunit salt bridge. The mutants R292D and D297R are totally inactive. The crystal structure of R292D reveals that the mutant enzyme retains the T-state quaternary structure. However, the mutation induces a reorganization of the interface with the creation of a network of interactions similar to that observed in the crystal structures of R-state yeast and M1 PK proteins. Furthermore, in the R292D structure, two loops that are part of the active site are disordered. The K382Q and R431E mutations were designed to probe the binding site for fructose 1,6-bisphosphate, the allosteric activator. R431E exhibits only slight changes in the regulatory properties. Conversely, K382Q displays a highly altered responsiveness to the activator, suggesting that Lys 382 is involved in both activator binding and allosteric transition mechanism. Taken together, these results support the notion that domain interfaces are critical for the allosteric transition. They couple changes in the tertiary and quaternary structures to alterations in the geometry of the fructose 1,6-bisphosphate and substrate binding sites. These site-directed mutagenesis data are discussed in the light of the molecular basis for the hereditary nonspherocytic hemolytic anemia, which is caused by mutations in human erythrocyte PK gene. ) for its activity. The reaction is essentially irreversible under physiological conditions and is critical for the control of the metabolic flux in the second part of glycolysis. Moreover, the substrate PEP and the product pyruvate are involved in a variety of metabolic pathways. Such a central position in the cellular metabolism is reflected in the regulatory properties of PK, which is a typical allosteric protein (1). The activity is controlled by several physiological effectors, including H ϩ , Mg 2ϩ

In Vivo and in Vitro Phosphorylation of Two Isoforms of Yeast Pyruvate Kinase by Protein Kinase A

“…Although affinity for FBP has not been measured, a suggestion of increase in affinity can be speculated from the n H of 2 displayed by the less phosphorylated Pyk1 in the presence of 1.5 mM FBP as compared with 1.4 for the more phosphorylated enzyme. A similar modification of the kinetic parameters has been described for a point mutation in the Escherichia coli pyruvate kinase (38) precisely in a residue (Arg-271) located in the interface between the catalytic domain A and the regulatory domain C of FBP binding, suggesting that Ser-22 in Pyk1, located structurally in the same interface, might be the target for PKA phosphorylation. It is interesting to mention that yeast cells, containing a point mutation in a residue (R19Q) located in the vicinity of Ser-22 (Arg-19, contained in the consensus PKA phosphorylation sequence RRTS) from yeast Pyk1, do not grow in glucose as carbon source (39), indicating null or low catalytic activity for the Pyk1 from this mutant.…”

MicroRNA-7 Promotes Glycolysis to Protect against 1-Methyl-4-phenylpyridinium-induced Cell Death

“…1 and 2). Pyruvate kinase activity is allosterically regulated by fructose 1,6-bisphosphate, P-enolpyruvate, and ATP (18,19). Although fructose 1,6-bisphosphate and P-enolpyruvate increase pyruvate kinase activity (19,20), high ATP level decreases it (19).…”

Fifteen novel mutations inPKLRassociated with pyruvate kinase (PK) deficiency: Structural implications of amino acid substitutions in PK

“…The C domain contains the binding site for FBP. Subunit interactions at the interfaces between the A domains (A/A 0 subunit interface) and the C domains (C/C 0 subunit interface), as well as A/B and A/C interdomain interactions within one subunit are considered to be key determinants of the allosteric response, which involves switching of the PK tetramer from the low-affinity T-state to the high-affinity R-state [Fenton and Blair, 2002;Jurica et al, 1998;Mattevi et al, 1995;Rigden et al, 1999;Valentini et al, 2000Valentini et al, , 2002Wooll et al, 2001].…”